Seeing a Neurotoxin's Deadly Grip

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Seeing a Neurotoxin's Deadly Grip

http://www.hhmi.org/news/botulinum20061213.html

Botulinum neurotoxin hijacks synaptic vesicle recycling at

neuromuscular junctions. The toxin first docks to the active zone

(blue) by binding to two membrane-anchored receptors, synaptotagmin

(red) and ganglioside (yellow). The toxin-receptor complexes are

then internalized by endocytosis.

Two Medical Institute research teams working

independently have discovered new information about how the

botulinum neurotoxin shuts down neurons with deadly efficiency. By

providing detailed views of the toxin plugged into its neuronal

receptor, the new studies could aid efforts to engineer specialized

versions of the powerful neurotoxin that is used to treat a wide

array of medical problems.

The two groups were led by HHMI investigators Axel Brunger at

Stanford University and Edwin Chapman at the University of Wisconsin

at Madison. They published their findings December 13, 2006, in

advance online publications in the journal Nature.

" These toxins could prove the ideal basis for drugs, because they

work at extremely low concentration. "

Axel T. Brunger

" Botulinum neurotoxins are powerful tools for biologists and find

widespread use as therapeutics for the treatment of certain nervous-

system diseases, " wrote Giampietro Schiavo of the London Research

Institute in an accompanying News & Views commentary in Nature. " For

these reasons, the papers reported here are of tremendous value. "

Botulinum neurotoxins are among the most deadly natural toxins in

the world. They act by first attaching themselves to receptors on

the surface of neurons. The toxins then insinuate an enzyme into the

neuron that degrades key proteins required for neurons to

communicate with one another. The toxins principally affect muscle-

controlling motor neurons activated by the neurotransmitter

acetylcholine. They kill by paralyzing the respiratory muscles.

There are seven structurally and functionally related botulinum

neurotoxins (BoNTs), called serotypes A through G, with each acting

in a slightly different manner. In 2004, Brunger's group published

an article in Nature detailing how the toxins that cause botulism

and tetanus can recognize and attack particular nerve cell proteins

at the neuromuscular junction.

Researchers knew that the toxins simultaneously bind to two distinct

neuronal receptors - one a protein and one a sugar-containing lipid

called ganglioside - but the details of that binding had not been

established prior to these studies.

Both research groups began by crystallizing the BoNT/B serotype

toxin in complex with its protein receptor, called synaptotagmin II.

Working with Chapman's group, co-authors s, Qing Chai

and ph Arndt of the Scripps Research Institute crystallized full

length BoNT/B in complex with the " recognition domain " of

synaptotagmin II, to which the toxin attaches. Simultaneously,

working in Brunger's group, lead author Rongsheng Jin, an HHMI

postdoctoral fellow, in collaboration with Binz and s

Rummel of the Medizinische Hochshule Hannover in Germany

crystallized the receptor-binding domain of BoNT/B in complex with

the recognition domain of synaptotagmin II, achieving a

significantly higher resolution of the complex.

Each research group determined the structure of the toxin-domain

complex using x-ray crystallography. In x-ray crystallography,

protein crystals are bombarded with intense beams of x-rays. As the

x-rays pass through and bounce off of atoms in the crystal, they

leave a diffraction pattern, which can then be analyzed to determine

the three-dimensional shape of the protein.

Both groups discovered that the toxin holds its receptor in an

intimate molecular embrace. The toxin induces a helix in the

synaptotagmin protein that fits precisely into a groove in the toxin

molecule. Both teams showed that they could disrupt this binding by

introducing mutations that would subtly alter the shape of the

synaptotagmin receptor.

Brunger and his colleagues found that altering the toxin at the

binding site by single amino acid changes (obtained from the high

resolution crystal structure) drastically reduced its toxicity.

Specifically, when they incubated the altered neurotoxin with mouse

diaphragm, it produced far less muscular paralysis than the natural

toxin.

" This tells us that it is possible to design a small-molecule

inhibitor that could powerfully disrupt the interaction between the

toxin and the receptor, " said Brunger. " Such inhibitors would act as

powerful, specific anti-toxins, with fewer side effects than current

drugs " Also, he said, detailed knowledge of toxin-receptor binding

could help in designing botulism vaccines. Such vaccines could

consist of fragments of protein corresponding to the toxin's binding

region, which could be used to trigger antibody production against

the toxin that would block its action.

Both research groups also explored the role of the ganglioside

binding site in the toxin's double-receptor binding. Brunger and his

colleagues found that the protein and ganglioside binding sites are

quite distinct, although they are both necessary for the toxin to

attach to the neuron and enter it.

Both groups found that docking between the toxin and the double

sites is more extensive than previously believed. " It's like

nestling a cube into a corner, " Chapman said. " The surfaces mesh

perfectly, so that the toxin is juxtaposed very near the membrane in

preparation for translocation into the cell. Practically speaking,

this knowledge of how the toxin recognizes the double receptor could

lead to a rational basis for designing small-molecule inhibitors to

this recognition step, " he said.

Brunger and Chapman emphasized that discoveries about the toxin-

receptor structure could lead to significant advances in engineering

the toxin for clinical application. Injection of the toxin is

already widely used to erase wrinkles and frown lines. However, the

toxin is also used to treat migraine headaches, involuntary

contraction of the eye muscles, overactive bladder syndrome,

excessive sweating and spastic disorders associated with injury or

disease. The researchers see the potential for even more widespread

use.

" These toxins could prove the ideal basis for drugs, because they

work at extremely low concentration, " said Brunger. " The therapeutic

doses are less than a nanogram per kilogram of body weight -

compared to milligram-per-kilogram concentrations needed for most

pharmaceutical drugs. "

" With knowledge of the toxin-receptor interaction, you can begin to

design toxin-receptor pairs to change the specificity of what cell

gets recognized, " said Chapman. " You can also change the specificity

of the protein the toxin cleaves inside the cell. " Thus, he said,

engineered toxins could provide targeted treatments for a wide array

of medical disorders. "

Courtesy of Axel Brunger, HHMI at Stanford University